Design of UCST Polymers for Chilling Capture of Proteins

Article pubs.acs.org/Biomac

Design of UCST Polymers for Chilling Capture of Proteins Naohiko Shimada, Miki Nakayama, Arihiro Kano, and Atsushi Maruyama* Institute for Materials Chemistry and Engineering, Kyushu University, Motooka 744-CE11, Nishi-ku, Fukuoka 819-0395, Japan S Supporting Information *

ABSTRACT: Ureido-derivatized polymers, such as poly(allylurea) (PU) and poly(L-citrulline) derivatives, exhibited upper critical solution temperature (UCST) behavior under physiological buffer conditions as we previously reported. The PU derivatives having amino groups (PUAm) also showed UCST behavior. In this study, we modified the amino groups of the polymer with succinyl anhydride (PU-Su) or acetyl anhydride (PU-Ac) to determine the effects of these ionic groups on the UCST behavior and to control interactions between the PU derivatives and biocomponents such as proteins and cells. Succinylation of PU-Am resulted in a significant decrease in phase separation temperature (Tp), whereas acetylation of PU-Am resulted in an increase in Tp. As expected, the Tp of PU-Am and PU-Su changed when the pH of the solution was changed. The Tp of PU-Am increased at higher pH, whereas that of PU-Su increased at lower pH, indicating that ionic charge decreases Tp of PU derivatives by increasing osmotic pressure and by increasing hydrophilicity of the polymer chains. Interestingly, these groups did not significantly change UCST when these groups were nonionic. We then examined capture and separation of particular proteins from a protein mixture by cooling-induced phase separation. Selective and rapid capture of particular proteins from protein mixture by PU derivatives was shown, indicating that the ureido-derivatized polymers are potential media for bioseparation under biofriendly conditions.

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INTRODUCTION There are many examples of polymers with lower critical solution temperature (LCST)-type thermoresponsive phase transition behaviors. These polymers are soluble below LCST and become insoluble above LCST. One of the most familiar examples of a thermoresponsive polymer is poly(N-isopropylacrylamide) (PNIPAM). Use of PNIPAM in biomedical and biotechnological technologies, such as drug delivery1,2 and tissue engineering3 devices, has been explored. The LCST of polymers can be controlled by introducing hydrophilic, hydrophobic, and ionic moieties. Conversion of thermoresponsiveness to pH responsiveness by introduction of pHsensitive ionic groups to LCST polymers was demonstrated.4−6 These polymers undergo phase transitions in response to pH7,8 and can be used for pH-triggered controlled release of drugs9 or pH-assisted drug targeting.10 Introduction of ionic groups also allows thermal control of the interactions of these polymers with proteins11,12 and cells.13 Polymer materials that are capable of responding to light,14 salt,15 or organic substances such as sugar16 have been prepared by modifying thermoresponsive polymers with appropriate functional groups. The thermoresponsive polymers that show opposite thermoresponsiveness, that is upper critical solution temperature (UCST) behavior, are also available. In general, UCST polymers exhibit thermoresponsiveness on the basis of cohesive interactions, such as hydrogen bonding or electrostatic interactions, between polymer chains that are destabilized at higher temperature.17 Hydrogen bonds and electrostatic interactions are stabilized in low polarity solvents. Thus, UCST behavior is rarely observed in aqueous media, especially © XXXX American Chemical Society

under physiological conditions, because both hydrogen bonds and electrostatic interactions between polymer chains are destabilized by water and salts. Recently, polymers with multiple amide groups were reported to exhibit UCST-type behavior in a phosphate buffer.18−20 Their utility in biomedical and biotechnological fields was, however, limited, because the phase separation temperatures of these polymers are lower than 30 °C. The low Tps of these polymers also restrict utility of chemical modification and functionalization of the polymers with ionic and hydrophilic groups because introduction of these groups usually results in a considerable decrease in Tp. Indeed, introduction of trace amounts of ionic groups as monomer impurities or initiator fragments to the polymers reduced or prevented the UCST-type behavior.21 It was suggested that, for UCST-type behavior in aqueous media, polymers must have groups with the ability to hydrogen bond and must have few ionizable groups. In support of this consideration, recently synthesized poly(acrylamide-co-acrylonitrile) and poly(methacrylamide) nonionic polymers have Tps above 37 °C.22 We previously reported that ureido-derivatized polymers, poly(allylurea) (PU) and poly(L-citrulline), exhibit UCST-type behavior under physiologically relevant pH and salt conditions.23 A UCST polymer with Tp > 60 °C was obtained by regulating ureido content and molecular weight (Mw). Because of the high Tp it is possible to introduce ionic amino groups up Received: January 24, 2013 Revised: February 26, 2013

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Figure 1. Structural formulas of (a) PU-Am, (b) PU-Su, and (c) PU-Ac. transmittance first began to drop. Tp values were reproducible with temperature differences less than 2 °C. Protein Capture/Separation Property of PU Derivatives. PUAm7 derivatives (final concentration: 1 mg/mL) dissolved in pure water were added to a 800 μg/mL solution of proteins (including βGal, Plb, BSA, OVA, CA, SBTI, Lys, and BPTI; 100 μg/mL each protein) in 10 mM HEPES/NaOH (pH 7.5) containing 50 mM NaCl (final volume: 100 μL). After incubation at 10 °C for 2 h or without incubation, the mixture was centrifuged at 4 °C for 10 min. The supernatant was analyzed by 15% SDS-PAGE. The bands were stained with SimplyBlue SafeStain (Invitrogen). After separation by PU-Am7, 100 μL of the buffer containing PVS (625 μM) was added to precipitate. The solution containing the precipitate or supernatant was diluted 40-fold by the same buffer. MUG (0.2 μM) as a substrate for βGal was added to the solutions at 37 °C, and fluorescence at 450 nm (λex 360 nm) was recorded for 200 s.

to 20 mol % without reducing Tp below the physiological range. The Tp of poly(allylurea-co-allylamine) (PU-Am, Figure 1a) could be controlled over the temperature range from 8 to 65 °C by changing amino group content. It was also possible to install pH responsiveness in phase transition behavior.23 In this study, we prepared succinylated PU-Am (PU-Su, Figure 1b) and acetylated PU-Am (PU-Ac, Figure 1c) to determine the effect of ionic groups on UCST-type behavior. We are also interested in control of interactions of the ureidoderivatized polymers with biocomponents such as proteins and cells through ionic interactions. We demonstrated that rapid and selective protein separation can be possible by employing PU having different ionic groups.

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EXPERIMENTAL SECTION

RESULTS AND DISCUSSION Phase Separation Behavior of PU Derivatives under Physiologically Relevant Conditions. The phase separation behaviors of PU derivatives were analyzed under physiologically relevant salt concentration and pH. A transmittance change at Tp = 44 °C was observed for PU-Am7, which has 7 mol % amino groups (Figure 2a, red line). Succinylation of PU-Am7 (PU-Su7) resulted in a 24 °C decrease in Tp (Figure 2a, blue line), whereas acetylation (PU-Ac7) did not significantly change the UCST (ΔTp = 3 °C, Figure 2a, black line). Thus, amino groups had much less effect on Tp than succinyl groups. The minor influence of amino group is also demonstrated by the small difference in Tp between poly(allylurea) homopolymer (Tp = 48 °C)23 and PU-Am7 (Tp = 44 °C). UCST behavior of these polymers occur over a relatively narrow temperature range of 4 °C. It was previously reported that the poly(N-acryloyl glycinamide) copolymer (PNAGA) with a small amount (5 mol %) of acrylic acid did not show UCST-type behavior in phosphate buffered saline (PBS), whereas the PNAGA homopolymer exhibited UCST-type behavior (Tp ∼ 10 °C) in PBS.21 In our study, PU derivatives containing 7 mol % of carboxylic or amino groups clearly exhibited UCST-type behavior under physiologically relevant conditions, suggesting that ureido polymers are potential starting materials for creating functionalized UCST polymers. To investigate more extensively the effect of ionic groups on UCST behavior of PU, we prepared PU-Am13 with approximately twice the amino group content (13 mol %) of PU-Am7. The phase transition occurred at lower temperature for PU-Am13 than for PU-Am7; the Δ Tp was 21 °C (Figure 2b, red line). The lower Tp of PU-Am13 than that of PU-Am7 is likely due to the higher amino group content and lower ureido group content. The amino groups were protonated

Materials. Poly(allylamine) hydrochloride (Mw: 1.5 × 104) was purchased from Nitto Boseki. Potassium cyanate, succinic anhydride, acetic anhydride, and bovine serum albumin (BSA) were purchased from Wako Pure Chemical Industries. β-Galactosidase from E. coli (βGal) was purchased from Funakoshi. Phosphorylase b from rabbit muscle (Plb), ovalbumin from chicken egg white (OVA), carbonic anhydrase from bovine erythrocytes (CA), soybean trypsin inhibitor (SBTI), lysozyme from chicken egg white (Lys), trypsin inhibitor from bovine pancreas (BPTI), 4-methylumbelliferone β-D-galactopyranoside (MUG), and poly(vinylsulfonic acid, sodium salt) (PVS) were purchased from Sigma-Aldrich. PU-Am7 and PU-Am13, which have 7 and 13 mol % amino residues, respectively, and 93 and 87 mol % ureido content, respectively, were prepared as previously described.23 Succinylated PU-Am (PU-Su7) and acetylated PU-Am (PU-Ac7) were synthesized by addition of 3 equiv (mole ratio to amino groups) of succinic anhydride or acetic anhydride to PU-Am in DMSO or water, respectively. The polymers were purified by dialysis against water and then lyophilized. The polymers in D2O containing 1% NaOD were characterized by 1H NMR (Bruker Avance 400). PU-Su: δ = 1.0−1.8 (polymer backbone, -CH2-CH-), 3.0 (-CH2-NH-CONH2), 2.4 ppm (succinyl, -NH-CO-CH2-CH2-COOH). PU-Ac: δ = 1.0−1.8 (polymer backbone, -CH2-CH-), 3.0 (-CH2-NH-CONH2), 2.4 ppm (acetyl, -CH2-NH-COCH3). The methylene signal (-CH2-NH2) at 2.6 ppm of PU-Am shifted to 3.0 ppm after the acetylation or succinylation, indicating that all amino groups of PU-Am were modified with acetyl or succinyl groups (Figure S1 of the Supporting Information). Transmittance Measurements. Stock solutions of polymers in pure water were diluted with pH-controlled buffer solutions containing various salts. Thermal change in transmittances at 500 nm of the polymer solutions in 10 mm quartz cells were measured on a Shimadzu UV-1650PC UV−visible spectrophotometer equipped with a Peltier temperature controller at scanning rate of 1 °C/min from 70 to 5 °C. The buffers used in this study were as follows: 10 mM acetic acid/NaOH (pH 4.5), 10 mM sodium cacodylate/HCl (pH 5.0), 10 mM MES/NaOH (pH 5.5 or 6.5), 10 mM HEPES/NaOH (pH 7.5 or 8.5), 10 mM borate/NaOH (pH 9.5 or 10.5) containing 150 mM NaCl. Tp was defined as an initial point of temperature where the B

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Figure 4. Tps of (a) PU-Am7 derivatives and (b) PU-Am13 derivatives as a function of pH. Buffers contained 150 mM NaCl.

Figure 2. Transmittance curves of (a) PU-Am7 derivatives and (b) PU-Am13 derivatives in 10 mM HEPES/NaOH (pH 7.5) containing 150 mM NaCl. Transmittances at 500 nm of PU-Am derivative solutions were measured at a scanning rate of 1 °C from 70 to 5 °C. The concentration of PU-Am derivatives was 1 mg/mL.

°C (Figure 2b, black line). The conversion of cationic amino groups to nonionic acetyl groups resulted in the increase in Tp. Succinylated PU-Am13 (PU-Su13) did not show the phase separation (Figure 2b, blue line). To estimate influence of carboxylate anions, the temperature responsiveness of PU-Su13 was evaluated in different salt conditions. No phase separation was observed in 150 mM NaCl, 150 mM KCl, or 150 mM NH4Cl (Figure 3). No phase separation was observed even when the NaCl concentration was increased to 1 M (data not shown). In the presence of 150 mM divalent cation, either Mg2+ or Ca2+, a phase transition of PU-Su13 was observed (Figure 3). These salt dependencies may be due to Debye screening of electrostatic interaction by the salt ions. Also, salt bridging of carboxyl groups with multivalent metal ions may be necessary for this increase of Tp. The Tp in Ca2+ was 20 °C higher than that in Mg2+ solution, suggesting potential of PUSu as cation-sensitive materials. pH Sensitivity in PU Derivatives. We next determined Tps of PU derivatives in solutions with pH ranging from 4.5, close to the pKa of a carboxyl group, to 10.5, close to the pKa of a protonated amino group (Figure 4). The Tps of PU-Ac7 and PU-Ac13, which have no ionizable groups, showed no pH dependence; it was approximately 41 and 35 °C at all pHs, respectively. This difference of Tp likely depends on ureido content. PU-Su series showed pH dependency at acidic pH. The Tps of PU-Su series increased with decreasing pH lower than 5.5, where ionic dissociation of their carboxyl groups was suppressed. Consistent with a previously published result,23

Figure 3. Effect of 150 mM indicated salt on UCST behavior of PUSu13 in 10 mM HEPES/NaOH (pH 7.5).

under the experimental pH, decreasing Tp of the polymers through osmotic and hydrophilic effects. Acetylation of amino groups of PU-Am13 (PU-Ac13) increased the Tp from 23 to 35 C

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Figure 5. Capture and separation of proteins by (a) PU-Am7, (b) PU-Su7, and (c) PU-Ac7. Mixtures containing 100 μg/mL each protein and PU derivatives were mixed and incubated at 10 °C for 2 h. After centrifugation of the solution, the supernatant was analyzed by 15% SDS-PAGE. Arrows indicate protein bands that disappeared or that were significantly reduced in intensity in the presence of PU derivatives.

Hence, 150 mM NaCl effectively screened ionic charges of PUAm7 . The higher Tps of PU-Am7 than those of PU-Ac may be explainable by the difference in salting out effect between amino and acetyl groups. These results indicated that ion charges along polymer chains significantly reduced Tps, and carboxylate anion showed stronger effect of decreasing Tp than ammonium cations. Ammonium cations and carboxylate anions equally contributed to osmotic pressure of polymer chains. The diverse effects of ammonium cations and carboxylate anions may be due to differences in hydrogen bonding interactions and/or affinities to couterions. Carboxylate anions are strong hydrogen bonding acceptors, whereas protonated amino groups act only as hydrogen bonding donors. Hence, the former may disturb the hydrogen-bonding network of ureido groups rich in hydrogen-bonding donors. Some effects due to steric hindrance by succinyl groups are also possible. Protein Capture and Separation by PU Derivatives. UCST-type polymers that undergo phase separation upon cooling are suitable media for bioseparations because thermal denaturation and damage to biocomponents can be minimized, compared to LCST-type polymer that needs heating for phase separation. To evaluate the potential for use of ureidoderivatized polymers as media for bioseparation, we carried out a capture/separation assay with a mixture of β-galactosidase (β-Gal), phosphorylase b (Plb), bovine serum albumin (BSA), ovalbumin (OVA), carbonic anhydrase (CA), soybean trypsin inhibitor (SBTI), lysozyme (Lys), and bovine pancreas trypsin inhibitor (BPTI). A solution of PU derivative dissolved in pure water was added to the protein mixture and incubated for 2 h at 10 °C, a temperature below the Tp of each polymer (PU-Am7: Tp = 34 °C, PU-Su7: Tp = 16 °C and PU-Ac7: Tp = 46 °C). Hence, upon the addition of derivative to the protein mixture, protein binding and phase separation would occur simultaneously. Figure 5 shows SDS-PAGE separations of supernatants of proteins obtained after centrifugation at 4 °C. Protein bands missing in lanes corresponding to the proteins that were captured by certain PU derivatives and precipitated. In the supernatant of PU-Am7/protein mixture, three bands corresponding to β-Gal (pI 5.3), Plb (pI 6.8), and SBTI (pI 4.5)

Figure 6. Selective capture and separation of proteins by PU-Am7. Proteins (100 μg/mL) were individually incubated with PU-Am7. (a) After centrifugation, supernatants were analyzed by 15% SDS-PAGE. (b) The amount of remaining protein was plotted as a function of PUAm7 concentration.

PU-Am13 showed pH dependency. Tp was increased at pH above 9, where deprotonation of amino groups took place. Interestingly, PU-Am7 did not show pH dependency. To investigate the observation, Tp of PU-Am7 was evaluated under lower salt conditions. In the absence of NaCl (Figure S2 of Supporting Information), Tp of PU-Am7 increased at pH > 9. D

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proteins likely influence affinity for the polymer derivatives. To further elucidate potentiality of PU derivatives as separation media, handling time was reduced as short as possible. PU derivatives and protein mixture were mixed by quick pipeting at 10 °C and immediately (within 1 min) centrifuged at 4 °C. As shown in Figure 7a, selective separation of proteins was also achieved. After the separation by PU-Am7, 92% activity of βGal was observed in precipitate, while the activity was slightly observed in the supernatant (Figure 7b). This result suggested that PU derivatives can quickly separate proteins keeping their activities. As described elsewhere,23 PU derivatives form hydrated coacervate droplets upon cooling. This phase transition property would be also advantageous for a separation media because hydrophobic interactions between separation media and proteins that caused nonspecific capture and protein denaturation were sufficiently weak. Furthermore, the liquid− liquid phase separation would allow rapid separation compared to conventional solid phase extractions using gel matrices or magnetic beads.

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CONCLUSIONS Cationic, anionic, and noncharged polymers exhibiting UCSTtype phase separation behavior under physiological relevant conditions were successfully prepared. These UCST polymers having ionic groups were sensitive to buffer conditions such as pH and salt conditions, indicating that charged state of the ionic groups strongly influence the UCST behavior. Cationic PU-Am7 selectively captured acidic proteins, whereas anionic PU-Su7 selectively captured basic proteins. PU-derivatives undergo soluble and hydrated coacervate states likely contributed the selective, rapid, and biofriendly separation. Amino or carboxyl groups of PU derivatives can be used as chemical modification sites with various ligands, such as antibodies, peptides, or drugs, for affinity separation of specific proteins. These polymers should have broad utilities as UCSTtype smart biomaterials for surface modification, gel scaffolds, and drug release and targeting matrices.

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Figure 7. (a) Rapid separation of proteins by PU derivatives. Mixtures containing 100 μg/mL each protein and PU derivatives were mixed (without incubation) at 10 °C. After centrifugation of the solution, the supernatant was analyzed by 15% SDS-PAGE. (b) Activities of β-Gal in precipitate and supernatant after separation by PU-Am7. MUG (0.2 μM) was added to solution containing the precipitate (solid red line), the supernatant (dotted red line), or protein mixture (2.5 μg/mL each protein; solid black line) at 37 °C, and fluorescence at 450 nm (λex 360 nm) was monitored.

ASSOCIATED CONTENT

S Supporting Information *

1

H NMR charts of PU-derivatives, transmittance curves of PUAm7 at various pH in the absence of NaCl, and SDS-PAGE analysis of protein capture and separation by PU-Am13. This material is available free of charge via the Internet at http:// pubs.acs.org.

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were missing (Figure 5a), indicating that PU-Am7 selectively captured these acidic proteins. Basic proteins such as Lys (pI 11.1) and BPTI (pI 10.5) were not captured by PU-Am7. Interestingly, acidic proteins BSA (pI 4.8), OVA (pI 4.6), and CA (pI 5.6) were not captured by PU-Am7, regardless of their low pI. Similar selective capture of proteins was observed for PU-Am13 (Figure S3 of Supporting Information). As shown by capture experiments with individual proteins as a function of PU-Am7 concentration, PU-Am7 has 3-fold higher affinity for β-Gal than for SBTI (Figure 6). In contrast to PU-Am7, PUSu7 selectively captured basic proteins Lys and BPTI (Figure 5b). PU-Ac7 showed very low affinity for the proteins tested, except for a modest affinity for Lys (Figure 5c). These results suggest that proteins are mainly captured through electrostatic interactions between ionic groups of proteins and functional groups of PU derivatives, although factors such as the threedimensional structures (surface distribution of amino acids) of

AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-92-802-2522. Fax: +81-92-802-2521. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Parts of this work were supported by a Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Nanomedicine Molecular Science’’ (No. 2306) by the Project of Integrated Research on Chemical Synthesis from the Ministry of Education, Culture, Sports, Science and Technology, by KAKENHI (No. 23240074), and by the SENTAN project from Japan Science and Technology Agency. E

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(21) Seuring, J.; Bayer, F. M.; Huber, K.; Agarwal, S. Upper critical solution temperature of poly(N-acryloyl glycinamide) in water: a concealed property. Macromolecules 2011, 45, 374−384. (22) Seuring, J.; Agarwal, S. First example of a universal and costeffective approach: polymers with tunable upper critical solution temperature in water and electrolyte solution. Macromolecules 2012, 45, 3910−3918. (23) Shimada, N.; Ino, H.; Maie, K.; Nakayama, M.; Kano, A.; Maruyama, A. Ureido-derivatized polymers based on both poly(allylurea) and poly(L-citrulline) exhibit UCST-type phase transition behavior under physiologically relevant conditions. Biomacromolecules 2011, 12, 3418−22.

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